The Hedgehog (Hh) family of secreted signaling molecules governs cell growth and patterning in numerous developmental processes in both vertebrates and invertebrates. Misregulation of Hh signaling activity has been implicated in many human disorders such as cancer. The goal of my laboratory is to understand how Hh signal is transduced to control cell growth and patterning. The Hh signal reception system consists of two transmembrane proteins: a multispan-transmembrane protein Patched (Ptc) and a seven-transmembrane protein Smoothened (Smo), whose mechanism of action remains a mystery. Hh binds and inhibitsPtc, leading to the activation of Smo. Smo then activates an intracellular signaling cascade that culminates at the activation of the latent transcription factor Cubitus interruptus (Ci).My lab has previously shown that protein kinase A (PKA) negatively regulates the Hh pathway by promoting Ci proteolysis to generate a truncated form with represser activity. Recently, we discovered that PKA also positively regulates the Hh pathway by phosphorylating Smo in response signal. This finding provides new tools and hypotheses to dissect the Hh signaling mechanism at the receptor level. In this proposal, we will focus on understanding how Smo is activated and how Smo activates immediate downstream components using a combination of genetic, cell biology, and biochemical approaches in both cell culture system and whole organism. Specifically, we will 1) investigate how phosphorylation regulates Smo cell surface expression and activity; 2) test the hypothesis that Smo dimerization modulates it signaling activity; 3) investigate the role of Smo/kinase interaction in Smo phosphorylation and action; 4) identify novel components regulating Smo cell surface accumulation and activity by protein-protein interaction, genetic interaction, and RNAi screens. The proposed study should provide novel insight into how Hh signal is transduced at the cell membrane: how Smo activity is regulated and how Smo functions. As unconstrained elevation of Smo activity contributes to many human cancers including skin, lung, pancreas, and prostate cancers, our study may provide new avenues for improving diagnosis and therapeutics of these cancers.